The presence of latent HIV in individuals treated with highly active combination antiretroviral therapy (cART) has been well established. Without additional therapies, individuals must remain on ART indefinitely in order to avoid development of severe immunodeficiency and spread of the virus. Because of the existence of this reservoir, treatment interruption invariably leads to viral rebound. Thus, it is generally accepted that eradication of the virus will require elimination of the latent reservoir. The fundamental goal of this proposal is adapt our primary TCM-based assay for HIV-1 latency described above to be amenable for testing of latency regulator genes via CRISPR/Cas9 technology. Our initial targets will therefore be candidate genes that are widely accepted to be important for either the establishment/maintenance of latency or for reactivation from latency, and a number of previously untested genes that were identified through genetic screens in various laboratories. Successful validation of novel genes/proteins that control viral latency will lead to identification of relevant pathways and mechanistic studies that will have the potential to spawn major new initiatives to develop novel therapeutics.